Affinity Particle And Method Of Affinity Separation

ABSTRACT

The present invention is affinity particles that are characterized by having phosphorylcholine groups represented by the following formula (1) covalently bonded onto the surface of organic particles and also by having ligands having specific affinity with a certain target substance covalently bonded or adsorbed onto the surface of organic particles. 
 
The object of the present invention is to provide an affinity separation method that uses affinity particles utilizing organic particles and is capable of separating the target substance easily and with high accuracy.

TECHNICAL FIELD

The present invention relates to affinity particles and an affinityseparation method. More specifically, it relates to affinity particlesutilizing organic particles and an affinity separation method thatallows easy and highly precise separation of the target substance. Theaffinity particles of the present invention are very useful in variousseparation, purification, and testing methods including latexagglutination methods and immunoprecipitation methods that allow easyand highly sensitive detection of the target substance.

BACKGROUND ART

Conventionally, column chromatography has been used for separation andpurification of biological substances. However, column separation hassome fatal problems as described in the following (1) to (3):

(1) Many kinds of columns have to be used to obtain the targetsubstance, resulting in a poor purification efficiency.

(2) A verification test is required to make sure the target substance iscontained in the fractionated ingredients, which means purification istime consuming.

(3) Because of the large purification loss, a large quantity of thesample is required.

On the other hand, for separation and purification of the targetsubstances, affinity particles and affinity columns supporting ligandsare used (Patent Document 1 and Patent Document 2).

However, separation and purification using affinity columns have thefollowing problems:

(1) The desired target substance is not selectively separated. That is,in addition to the target substance captured by the ligand, unwantedsubstances are also adsorbed onto the column.

(2) The capture efficiency is low, which means a large quantity of theliquid sample is required.

The affinity separation or method in which affinity particles aredispersed in a liquid sample for separation uses agarose and such(Non-patent Document 1), but this method has a problem in that thedesired target substance is not selectively separated. That is, inaddition to the target substance captured by the ligand, unwantedsubstances are also adsorbed onto the affinity particles.

In addition to the aforementioned fatal problems, the affinity particlesmade of organic particles have a problem in that the organic particlestend to aggregate in samples having a high salt concentration. Becauseof this, the measurement has to be conducted with a diluted sample.

Patent Document 1: Japanese Patent Publication H8-26076

Patent Document 2: Japanese Patent Laid-Open H2002-511141 bulletin

Non-patent Document 1: Bioconjugate Chem.; 2002; 13(2); 163-166

DISCLOSURE OF INVENTION Problem that the Present Invention Aims to Solve

The present invention aims to solve the aforementioned problems andprovides affinity particles composed of organic particles that are usedfor various separation, purification, and/or testing methods.

MEANS TO SOLVE THE PROBLEM

That is, the present invention provides affinity particles that arecharacterized by having phosphorylcholine groups represented by thefollowing formula (1) covalently bonded onto the surface of organicparticles.

Also, the present invention provides affinity particles that arecharacterized by having phosphorylcholine groups represented by thefollowing formula (1) covalently bonded onto the surface of organicparticles and also by having reactive groups or adsorptive groups, whichare capable of bonding with ligands having specific affinity with acertain target substance, covalently bonded or adsorbed onto the surfaceof organic particles.

Furthermore, the present invention provides affinity particles that arecharacterized by having phosphorylcholine groups represented by thefollowing formula (1) covalently bonded onto the surface of organicparticles and also by having ligands having specific affinity with acertain target substance covalently bonded or adsorbed onto the surfaceof organic particles.

Also, the present invention provides the aforementioned affinityparticles wherein said organic particles are either synthetic particleswhose polymers contain one, two, or more types of monomer units chosenfrom a group consisting of styrene, glycidyl methacrylate, (meth)acrylicacid, N-alkylacrylamide, and alkyl (meth)acrylate, or polysaccharidescomposed of agarose or sepharose having an average particle size of 20nm to 500 μm.

Furthermore, the present invention provides the aforementioned affinityparticles wherein said ligands are one, two, or more types of ligandschosen from a group consisting of various antibodies, antigens, enzymes,substrates, receptors, peptides, DNA, RNA, aptamers, protein A, proteinG, avidin, biotin, chelating compounds, and various metal ions.

Also, the present invention provides a method of affinity separation ofa target substance by using organic particles that includes

(1) a first process whereby arbitrary ligands are bonded to the affinityparticles of claim 1 or 2,

(2) a second process whereby the affinity particles prepared in thefirst process are dispersed in a liquid sample containing a targetsubstance selectively captured by the arbitrary ligands, and (3) a thirdprocess whereby the target substance captured is recovered from theaffinity particles.

Furthermore, the present invention provides a method of affinityseparation of a target substance by using organic particles thatincludes (1) a first process whereby the affinity particles of claim 3are dispersed in a liquid sample containing a target substanceselectively captured by the arbitrary ligands, and (2) a second processwhereby the target substance captured is recovered from the affinityparticles.

When the affinity particles of the present invention are used fordetection of antibodies and protein, such as in the immunoprecipitationmethod and the latex agglutination method, the recovery process (2) isnot required; detection can be done easily by visually observing changesin the dispersion state.

EFFECTS OF THE INVENTION

The affinity particles of the present invention use ligands to captureonly a certain target substance (the substance desired to be separated)and suppresses adsorption of other substances onto the particles,resulting in a very high separation selectivity. Also, due to theirsuperior dispersion properties, the target substance can be easily andaccurately separated without causing aggregation even in a sample havingvarious salts, such as serum.

That is, the target substance separation method of the present inventioncan effectively and easily separate the target substance to be separatedin a short amount of time. Since substances have a tendency to adsorbonto foreign substances, conventional affinity particles havedifficulties efficiently isolating only the target substance; however,it is possible to very efficiently prevent non-specific adsorption ofthe target substance to the affinity particles and thus increase thepurification yield by modifying the particle surface withphosphorylcholine groups.

Also, phosphorylcholine groups are extremely hydrophilic and they alsoimprove the dispersion properties of the affinity particles in a liquidsample containing water.

Furthermore, conventional particles tend to aggregate in the presence ofsalts and therefore the purification efficiency decreases when thetarget substance is to be isolated from serum because of aggregation dueto various salts in serum; however, the affinity particles of thepresent invention don't aggregate significantly even under the presenceof salts, which makes it possible to recover the target substanceefficiently.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic showing the difference between the protein captureselectivity of the affinity particles of the present invention andconventional affinity particles.

FIG. 2 shows a chemical structure formula and an NMR spectrum of thechemical compound prepared in Synthesis example 1.

FIG. 3 shows a chemical structure formula and an NMR spectrum of thechemical compound prepared in Synthesis example 2.

FIG. 4 shows a graph comparing the particle size distribution ofconventional affinity particles in water and in a saline solution.

FIG. 5 shows a graph comparing the particle size distribution of theaffinity particles of the present invention in water and in a salinesolution.

FIG. 1 is a graph comparing the protein adsorption level of thestyrene-glycidyl methacrylate particles and PC particles (A) prepared inReference example 1.

FIG. 7 is a graph comparing the protein adsorption level of thestyrene-glycidyl methacrylate particles and PC particles (B) and (C)prepared in Reference example 2.

FIG. 8 is a graph comparing the protein adsorption level of the agarosebeads and PC particles (D) prepared in Reference example 3.

FIG. 9 is a graph comparing the antibody selectivity of the affinityparticles of Example 1.

FIG. 10 is a graph comparing the antibody selectivity of the affinityparticles of Comparative example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail below.

“Organic Particles”

The selection of the organic particles that constitute the affinityparticles is not limited in particular in the present invention. “Anorganic particle” generally means any organic object having an averageparticle size of about 20 nm to 500 μm. Specific examples of suchparticles include synthetic particles whose polymer contains one, two ormore types of monomer units chosen from a group consisting of styrene,glycidyl methacrylate, (meth)acrylic acid, N-alkylacrylamide, alkyl(meth)acrylate, aminoalkyl (meth)acrylate, and hydroxyalkyl(meth)acrylate, or organic particles composed of agarose or sepharose.Hybrid particles having a core-shell structure in which the outer layeris organic and the inner particle is inorganic are also included.

Particularly preferable particles are those easily synthesized by meansof emulsion polymerization, suspension polymerization, etc.; examplesinclude styrene-divinylbenzene copolymer, styrene-glycidylmethacrylate-divinylbenzene copolymer, acrylic acid-N-isopropylacrylamide-methylene bisacrylamide copolymer, 2-hydroxymethacrylate-styrene-divinylbenzene copolymer, and 2-aminoethylmethacrylate-N-isopropyl acrylamide-methylene bisacrylamide copolymer.

Since the phosphorylcholine group represented by the aforementionedformula (1) and reactive groups or adsorptive groups that are capable ofbonding with ligands are to be introduced onto the particle surface bymeans of covalent bonding, the surface should preferably have reactivegroups such as amino groups, carboxyl groups, hydroxyl groups, and thiolgroups.

Also, the affinity particles having an average particle size of theorganic particles of 20 nanometers to 500 μm are preferable.

Examples include styrene-divinylbenzene copolymer, styrene-glycidylmethacrylate-divinylbenzene copolymer, acrylic acid-N-isopropylacrylamide-methylene bisacrylamide copolymer, 2-hydroxymethacrylate-styrene-divinylbenzene copolymer, and 2-aminoethylmethacrylate-N-isopropyl acrylamide-methylene bisacrylamide copolymer.

“Reactive Groups or Adsorptive Groups to which the Ligand can Bind”

The selection is not limited as long as bonding with the ligand ispossible. Preferable examples of the covalent bond form include anamide, ester, urethane, ether, secondary amine, urea bond, and disulfidebond. Therefore, reactive groups for which ligands can take thecorresponding covalent bond forms are preferable; examples include anamino group, hydroxyl group, carboxyl group, thiol group, etc. Also, forthe adsorption form, preferable are an avidin-biotin, metal-chelatingcompound, etc. Therefore, adsorptive groups for which ligands can takethe corresponding adsorptive forms are preferable; examples includeavidin, biotin, and chelating compounds.

“Ligands”

In the present invention, a “ligand” means a substance that bindsspecifically to a certain target substance; examples include variousantibodies, antigens, enzymes, substrates, receptors, peptides,aptamers, protein A, protein G, avidin, biotin, chelating compounds, andvarious metal ions Examples of the various antibodies include IgG, IgM,IgA, IgD, IgE, IgY, and polysaccharides, examples of enzymes includeglutathione-S-transferase, examples of substrates include glutathione,examples of receptors include hormone receptors, cytokine receptors,examples of ligands include lectin, examples of chelating compoundsinclude nitrile triacetate, and examples of various metal ions includeNi²⁺, Co²⁺, Cu²⁺, Zn²⁺, and Fe³⁺.

“A Method of Preparing the Affinity Particles of the Present Inventions”

Since the essence of the present invention is to have thephosphorylcholine group represented by formula (1) covalently bonded onthe surface of organic particles and also for the organic particles tohave reactive groups or adsorptive groups capable of bonding to ligandshaving specific affinity to a certain target substance that are directlypresent on their surface by means of covalent bonding or adsorption,there is no limitation on the selection of the preparation method;bonding can be done with any means.

However, as mentioned earlier, this does not include methods in which apolymer already having the phosphorylcholine group and reactive groupsor adsorptive groups capable of bonding to ligands is used to simplycoat the particle surface without chemical bonding. This is because thecoating polymer can peel off and/or there may be an influence from thecoating polymer.

The affinity particles of the present invention can be prepared with thefollowing method, for example.

Step 1: The phosphorylcholine group represented by the following formula(1) and reactive groups or adsorptive groups capable of bonding toLigands are introduced onto the particles. The selection of the reactivegroup or adsorptive group is not limited; examples include an aminogroup, hydroxyl group, carboxyl group, and thiol group.

Step 2: The phosphorylcholine group represented by formula (1) and theligand are bonded to the reactive group or adsorptive group introducedonto the particles. Any chemical structure (spacer) can exist betweenthe phosphorylcholine group or ligand and the reactive group oradsorptive group. Examples of such arbitrary spacers include a methylenechain, oxyethylene chain, as well as an alkylene chain containing one ora plurality of amino groups.

“When the Reactive Group or Adsorptive Group on the Particle Surface isan Amino Group”

Step 1: Amino groups are introduced to any particle by using a prior artmethod or a method that will be developed in the future. Amino groupsare directly introduced onto the particle surface. The amino group canbe a primary amine or a secondary amine.

Step 2: An aldehyde derivative or hydrate derivative obtained by theoxidative cleavage reaction of glycerophosphorylcholine is used in areductive amination reaction to directly add phosphorylcholine groups tothe surface of the particle having amino groups.

Not all the amino groups are bonded with the phosphorylcholine group(the reaction level is controlled) so that the remaining amino groupsare available as substituents for the ligand to bind to.

Or, a carboxyl derivative obtained by the oxidative cleavage ofglycerophosphorylcholine is used in an amidation reaction to directlyadd phosphorylcholine groups to the surface of the particles havingamino groups. Not all the amino groups are bonded with thephosphorylcholine group (the reaction level is controlled) so that theremaining amino groups are available as substituents for the ligand tobind to.

“A Method of Introducing Amino Groups onto the Particle Surface”

Examples of a prior art method for introducing amino groups to theparticles (step 1) follow:

1. Introduction of Amino Groups by Means of a Surface Reaction via aPlasma Treatment

Amino groups are introduced to the particle surface by means of a lowtemperature plasma in a nitrogen gas atmosphere. Specifically, theparticles are put into a plasma reactor vessel and, after a vacuum pumpis used to form a vacuum in the reactor vessel, nitrogen gas andhydrogen gas are introduced. Amino groups can be then introduced ontothe particle surface by means of glow discharge. It is also possible tomechanically turn the plasma-treated organic material into particles.References related to the plasma treatment are shown below:

1. M. Muller, C. oehr

Plasma aminofunctionalisation of PVDF microfiltration membranes:comparison of the in plasma modifications with a grafting method usingESCA and an amino-selective fluorescent probe Surface and CoatingsTechnology 116-119 (1999) 802-807

2. Lidija Tusek, Mirko Nitschke, Carsten Werner, Karin Stana-Kleinschek,Volker Ribitsch Surface characterization of NH3 plasma treated polyamide6 foils

Colloids and Surfaces A: Physicochem. Eng. Aspects 195 (2001) 81-95

3. Fabienne Poncin-Epaillard, Jean-Claude Brosse, Thierry Falher

Reactivity of surface groups formed onto a plasma treated poly(propylene) film

Macromol. Chem. Phys. 200. 989-996 (1999)

2. Introduction of amino groups by means of a surface modifier

The surface of the organic particles such as alkoxysilylgroup-containing particles is treated with a surface modifier havingamino groups, such as alkoxysilane, chlorosilane, and silazane.

For example, alkoxysilyl group-containing particles are treated with3-aminopropyltrimethoxysilane, which has a primary amino group, tointroduce amino groups. Specifically, the3-trimethoxysilylpropyl-1-methacrylate-methylmethacrylate-divinylbenzene copolymer particles are soaked in a mixedsolution of water and 2-propanol, and, after adding3-aminopropyltrimethoxysilane, the temperature is raised to 50° C. andthe reaction is carried out for six hours. After cooling down to roomtemperature, the aforementioned polymer is rinsed with methanol anddried to obtain particles that have amino groups directly introducedonto the aforementioned copolymer particles.

3. Introduction of amino groups by means of the silicone vapor phasetreatment (Refer to Japanese Patent Publication No. H1-54379, JapanesePatent Publication No. H1-54380 bulletin, and Japanese PatentPublication No. H1-54381 bulletin.)

The particle surface is treated with1,3,5,7-tetramethylcyclotetrasiloxane and then Si—H groups introducedonto the surface are reacted with monomers having an amino group toobtain an aminated surface. For example, styrene-divinylbenzeneparticles and 1,3,5,7-tetramethylcyclotetrasiloxane are put into adesiccator and an aspirator is used to deaerate it. The reaction iscarried out for 16 hours at 80° C., and the aforementioned particles aretaken out and dried at 50° C. The obtained particles are dispersed inethanol, to which allylamine is added, and an ethanol solution ofchloroplatinic acid is added, followed by two hours of stirring at 60°C. After the reaction is completed, filtration, ethanol rinsing, andreduced-pressure drying are carried out to obtain aminated organicparticles.

For the monomer to be used in this method, an amine-type monomer can beused. The amine-type monomer is not limited to allylamine as long as ithas a reactive site such as polymerizable vinyl and acrylate, and anamino group. The amino group can be protected by a butoxycarbonyl group,benzyloxycarbonyl group or the like.

In addition to an amine-type monomer, a monomer having a functionalgroup such as an epoxy group, to which an amino group can be easilyintroduced by means of, for example, a reaction with diamine, can beused as well.

“A Method for Introducing Phosphorylcholine Groups onto the Particleshaving Amino Groups”

Next, a method for introducing phosphorylcholine groups onto theaminated particle surface (step 2) is described below.

The particles are soaked in methanol, to whichphosphatidylglyceroaldehyde is added, and [the mixture is] left alonefor six hours at room temperature. Sodium cyanoborate is then added at0° C., followed by overnight heating and stirring, to add aphosphorylcholine group to an amino group. The particles are rinsed withmethanol and dried to obtain particles that have phosphorylcholinegroups directly on the surface. For the reaction solvent, proticsolvents such as water, ethanol, and 2-propanol can be used in additionto methanol; the introduction rate tends to be higher when methanol isused.

Or, the particles are dispersed in a mixed solution ofdimethylsulfoxide-water, to which N-hydroxysuccinimide,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, andcarboxymethyl phosphorylcholine dissolved in a mixed solution ofdimethylsulfoxide-water is added. After stirring for 6 hours at roomtemperature the particles are thoroughly rinsed with water and then theyare dried to obtain particles having phosphorylcholine groups directlyon the surface. In addition to what is mentioned above, aprotic solventssuch as N,N′-dimethylformamide, tetrahydrofuran, and acetonitrile can beused as the reaction solvent. Or, carboxymethyl phosphorylcholine andthionyl chloride are reacted and the obtained acid chloride is reactedwith the particles under anhydrous conditions using a solvent such asN,N′-dimethylformamide and acetonitrile, the particles are thenthoroughly rinsed with water and dried to obtain particles havingphosphorylcholine groups directly on the surface. This method alsoallows an efficient reaction with hydroxyl groups on the surface, andtherefore is effective when particles are composed of a polysaccharidesuch as agarose and sepharose or 2-hydroxyethyl (meth)acrylate.

Shown below is a scheme in which amino groups are introduced ontoorganic particles having alkoxysilyl groups by using 3-aminopropyltrimethoxysilane as the surface modifier and then phosphorylcholinegroups (abbreviated as PC) are introduced.

As described above, the particles directly having phosphorylcholinegroups on the surface can be obtained by a method in which particleshaving amino groups are prepared and then a reductive amination reactionwith a hydrate derivative or aldehyde derivative obtained by theoxidative cleavage reaction of glycerophosphorylcholine is used todirectly add phosphorylcholine groups to the particle surface.

This method has the following great advantages: the introduction rate ofthe phosphorylcholine group is high, and the surface of various organicparticles can be modified.

In the aforementioned method, the compound containing the aldehydederivative obtained by the oxidative cleavage reaction ofglycerophosphorylcholine is obtained by oxidative cleavage of the priorart glycerophosphorylcholine group by means of a prior art method, whichis a very easy step. This reaction uses periodic acid or periodate tooxidize 1,2-diol to open the bond and obtain two aldehyde derivatives;in this particular method, a phosphorylcholine aldehyde derivative andformaldehyde are produced. The reaction is usually carried out in wateror in an organic solvent containing water. The reaction temperature isbetween 0° C. and room temperature. The aldehyde derivative may gothrough an equilibrium reaction in water to become a hydrate, but thisdoes not affect the subsequent reaction with the amine. A scheme forpreparing a monofunctional aldehyde derivative containing aphosphorylcholine group is described below.

The reductive amination reaction for bonding the aldehyde derivative (orhydrate derivative) obtained by the oxidative cleavage reaction ofglycerophosphorylcholine to the amino groups of the particles can becarried out easily by stirring both of them in a solvent. This reactionis carried out by dissolving or dispersing these two in water or alcohol(a third organic solvent ingredient can be mixed in, too) to form animine and reducing it with a reducing agent to obtain a secondary amine.For the reducing agent, a mild reducing agent such as sodiumcyanoboronate is preferable, but other reducing agents can be used aslong as the phosphorylcholine is stable. The reaction is usually carriedout at 0° C. to room temperature, but heating may be added depending onthe situation.

It is also possible to react any amount of the compound represented byformula (2) to the aforementioned amino groups and leave the remainingamino groups as reactive groups or adsorptive groups to which ligandscan bind.

n denotes an integer 1-12.

“Reactive Groups or Adsorptive Groups to which Ligands can Bind”

Not all the amino groups are bonded with the phosphorylcholine group(the reaction level is controlled) so that the remaining amino groupsare available as reactive groups or adsorptive groups for the ligand tobind to. These particles are the affinity particles described in claim2, i.e. the particles that are organic particles directly having ontheir surface the phosphorylcholine groups represented by formula (1)and reactive groups or adsorptive groups to which the ligand can bind.When the ligand is bonded to these remaining amino groups, the affinityparticles described in claim 3, i.e. the particles directly having thephosphorylcholine group represented by formula (1) and the ligand ontheir surface, are obtained.

The product form of the affinity particles described in claim 2 is suchthat the user can bind any ligand to the particles depending on thesubstance to be captured (target substance). The product form of theaffinity particles described in claim 3 is such that the ligand isalready bonded. The affinity particles described in claim 1 are affinityparticles having at least the phosphorylcholine group of formula (1) onthe particle surface and their product form is such that the user canbind any ligand to them depending on the substance to be captured(target substance), regardless of the presence or absence of the ligandor reactive group or adsorptive group that can bind to it. Affinityparticles of any form are included as long as the phosphorylcholinegroup of formula (1) is present on the particle surface; for example,the forms described in claim 2 and claim 3 are included as well.

In the aforementioned reaction, leaving some amino groups as reactivegroups or adsorptive groups to which the ligand can bind can be madepossible, for example, by adjusting the reaction quantity or by acompetitive reaction of 3-aminopropyl trimethoxysilane and 3-aminopropyltrimethoxysilane to which the phosphorylcholine group is introduced.

It is also possible to react this amino group with a compound having anyfunctional group and use this functional group as the reactive group oradsorptive group to which the ligand can bind. Examples includeglutaraldehyde, alkyl diimidate, acyl azides, and isocyanates.

In a scheme in which 3-aminopropyl trimethoxysilane is used for theaforementioned surface modifier, it is also possible to adjust thereaction quantity of the surface modifier to leave some hydroxyl groups(OH) on the particle surface and use these remaining OH groups asreactive groups or adsorptive groups to which the ligand can bind.

“A Method of Binding the Ligand to the Particles having Amino Groups”

When the ligand is a protein, one aldehyde group of glutaraldehyde isreacted with an amino group on the organic particle and the otheraldehyde group is reacted with an amino group in the protein, thusbinding the protein.

“When the Reactive Group or Adsorptive Group on the Particle Surface isa Hydroxyl Group”

When hydroxyl groups are present on the organic particles, no reactivegroup or adsorptive group to which the ligand can bind, such as aminogroups as mentioned above, needs to be introduced; the hydroxyl groups(OH) present on the particle surface are used as they are to introducethe phosphorylcholine group and the ligand or reactive groups oradsorptive groups to which the ligand can bind. The affinity particlesof the present invention are preferably prepared with this method.

“A Method for Introducing Phosphorylcholine Groups onto the Particleshaving Hydroxyl Groups”

A chemical bond is formed by dehydration of the hydroxyl group on theparticle surface and Si—OMe of the compound of the following formula (3)or (4). This chemical reaction proceeds very easily and quantitativelyin most organic solvents if heating and refluxing are provided.Chemically and physically very stable phosphorylcholine groups can beintroduced by means of this dehydration reaction, which is preferable.The phosphorylcholine group-containing compound represented by thefollowing formula (3) or (4) is a new compound.

In this formula, m denotes 2-6 and n denotes 1-4. OMe can be replaced byOEt or Cl. Up to two of the OMe's, OEt's, or Cl's to be bonded to Si canbe replaced by a methyl group, ethyl group, propyl group, isopropylgroup, or isobutyl group.

“A Method of Preparing the Phosphorylcholine Group-Containing ChemicalCompound of Formula (3)”

The phosphorylcholine derivative shown in the following formula (5) isdissolved in distilled water. The phosphorylcholine derivative of thefollowing formula (5) is a prior art chemical compound and commerciallyavailable.

An aqueous solution of the chemical compound of formula (5) is cooled inan ice water bath; then sodium periodate is added, followed by fivehours of stirring. The reaction fluid is concentrated under reducedpressure and dried under reduced pressure; methanol is used to extract aphosphorylcholine derivative having an aldehyde group shown in thefollowing formula (6).

0.5 equivalents of 3-aminopropyltrimethoxysilane is added to themethanol solution of formula (6). This mixed solution is stirred for aprescribed amount of time at room temperature and cooled with ice; anappropriate amount of sodium cyanohydroborate is then added and thetemperature is returned back to room temperature, followed by 16 hoursof stirring. During this time dry nitrogen is continued to be fedthrough the reaction vessel. After filtering the precipitate, a methanolsolution of formula (3) is obtained.

The procedure described above can be carried out in the same way evenwhen m and n in the chemical compounds represented by formula (3)change. The procedure shown here is for m=3 and n=2. The reactionsolvent is not limited in particular; in addition to methanol, which wasmentioned above, water, alcohols such as ethanol, propanol, and butanol,and aprotic solvents such as DMF and DMSO can be used. Dehydratedsolvents are preferable to prevent polymerization during the reaction;of these, dehydrated methanol is particularly preferable.

If a methoxy group (OMe) in formula (3) is replaced by an ethoxy group(OEt), then the reaction is carried out by using ethanol instead ofmethanol; if it is replaced by C, then dimethylformamide ordimethylsulfoxide is used instead.

Furthermore, even when one or two of the OMe groups, OEt, or Cl's to bebonded to Si are replaced by a methyl group, ethyl group, propyl group,isopropyl group, or isobutyl group, the preparation can be carried outin exactly the same manner as described above.

“A Method of Preparing the Phosphorylcholine Group-Containing ChemicalCompound of Formula (4)”

An aqueous solution of the chemical compound of formula (5) is cooled inan ice water bath; sodium periodate and a catalytic amount of rutheniumtrichloride are added, followed by three hours of stirring. The reactionfluid is concentrated under reduced pressure and dried under reducedpressure; methanol is used to extract a phosphorylcholine derivative (7)having a carboxyl group.

Next, 1.2 equivalents of thionyl chloride is added to formula (7)dispersed in acetonitrile or N,N-dimethylformamide and, after 30 minutesof stirring, 0.9 equivalents of 3-aminopropyltrimethoxysilane is addedto the solution. This mixed solution is stirred for 4 hours at roomtemperature to obtain the chemical compound of formula (8).

In addition to thionyl chloride, any reagent can be used for theaforementioned condensation reaction as long as it generates halogenatedcarboxylic acid; examples include phosphorus pentachloride, phosphorusoxychloride, phosphorus tribromide, and oxalyl chloride.

In addition to using the silane coupler of formula (8), it is alsopossible to have the compound of formula (7) directly react with thehydroxyl group. For example, sepharose beads are dispersed in anhydrousacetonitrile, to which an acetonitrile solution prepared by mixing thecompound of formula (7) and 1.2 equivalents thionyl chloride, followedby overnight stirring, is added; after 3 hours of stirring, particleshaving the phosphorylcholine compound on the surface are obtained.

“Reactive Groups or Adsorptive Groups to Which Ligands can Bond”

Not all the hydroxyl groups are bonded with the phosphorylcholine group(the reaction level is controlled) so that the remaining hydroxyl groupsare available as reactive groups or adsorptive groups for the ligand tobind to. These particles are the affinity particles described in claim2, i.e. the particles that are organic particles directly having ontheir surface the phosphorylcholine groups represented by formula (1)and reactive groups or adsorptive groups to which the ligand can bond.When the ligand is bonded to these remaining hydroxyl groups, theaffinity particles described in claim 3, i.e. the particles directlyhaving the phosphorylcholine group represented by formula (1) and theligand on their surface, are obtained.

The product form of the affinity particles described in claim 2 is suchthat the user can bind any ligand to them depending on the substance tobe captured (target substance). The product form of the affinityparticles described in claim 3 is such that the ligand is alreadybonded. The affinity particles described in claim 1 are affinityparticles having at least the phosphorylcholine group of formula (1) onthe particle surface and their product form is such that the user canbind any ligand to them depending on the substance to be captured(target substance), regardless of the presence or absence of the ligandor reactive group or adsorptive group that can bind to the ligand.Affinity particles of any form are included as long as thephosphorylcholine group of formula (1) is present on the particlesurface; for example, the forms described in claim 2 and claim 3 areincluded as well.

“A Method of Binding the Ligand to the Particles having Hydroxyl Groups”

When the ligand is a protein, hydroxyl groups on the particles areactivated by using cyanogen bromide. Amino groups in the protein arereacted to these to bind the protein.

It is also possible to react this hydroxyl group with a compound havingany functional group and use this functional group as the reactive groupor adsorptive group to which the protein can bind.

“When the Reactive Group or Adsorptive Group on the Particle Surface isa Carboxyl Group”

Step 1: Carboxyl groups are introduced to any particle by using a priorart method or a method that will be developed in the future. Carboxylgroups are directly introduced onto the particle surface.

Step 2: It is also possible to react the phosphorylcholine-containingcompound represented by formula (9) with the particles having carboxylgroups so as to form an acid amide bonding with the phosphorylcholinegroup and use the remaining carboxyl groups as reactive groups oradsorptive groups to which ligands can bind.

Not all the carboxyl groups are bonded with the phosphorylcholine group(the reaction level is controlled) so that the remaining carboxyl groupsare available as reactive groups or adsorptive groups for the ligand tobind to.

“A Method of Introducing Carboxyl Groups onto the Particle Surface”

Examples of a prior art method for introducing carboxyl groups to theparticles (step 1) follow:

1. Introduction of carboxyl groups by means of a surface modifier

The surface of the organic particles such as alkoxysilylgroup-containing particles is treated with a surface modifier havingcarboxyl groups, such as alkoxysilane, chlorosilane, and silazane.

For example, organic particles having alkoxysilyl groups are treatedwith triethoxysilylpropyl succinate anhydrate to introduce carboxylgroups. Specifically, triethoxysilylpropyl succinate anhydrate isdissolved in N,N-dimethylformamide, to which distilled water and4-dimethylaminopyridine is added, followed by stirring at roomtemperature for 16 hours to obtain a silane coupling agent havingcarboxylic acid. This reaction is a hydrolysis reaction of succinic acidanhydrate using 4-dimethylaminopyridine.

Organic particles having alkoxysilyl groups are soaked in awater-2-propanol mixed solution, to which the silane coupling agenthaving carboxylic acid is added, followed by heating up to 50° C. for 6hours of reaction. After cooling down to room temperature, the organicparticles are rinsed with methanol and dried to obtain particles thathave carboxyl groups directly introduced onto organic particles.

2. Introduction of carboxyl groups by means of the silicone vapor phasetreatment (Refer to Japanese Patent Publication No. H1-54379, JapanesePatent Publication No. H1-54380 bulletin, and Japanese PatentPublication No. H1-54381 bulletin.)

The particle surface is treated with1,3,5,7-tetramethylcyclotetrasiloxane and then Si—H groups introducedonto the surface are reacted with monomers having a carboxyl group toobtain a carboxylated surface.

For the monomer to be used in this method, a carboxyl-type monomer canbe used. The selection of the carboxyl-type monomer is not limited aslong as it has a reactive site such as a carboxyl group, polymerizablevinyl and acryl.

“A Method for Introducing Phosphorylcholine Groups onto the Particleshaving Carboxyl Groups”

Next, a method for introducing phosphorylcholine groups onto thecarboxylated particle surface (step 2) is described below.

When particles having carboxyl groups on the surface are soaked in asolution of N-hydroxysuccinimide and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, the particle surface iscoated with active ester groups. A solution of the phosphorylcholinederivative having an amino group represented by formula (9) is added tothis to introduce phosphorylcholine groups.

“Reactive Groups or Adsorptive Groups to which Ligands can Bind”

Not all the carboxyl groups are bonded with the phosphorylcholine group(the reaction level is controlled) so that the remaining carboxyl groupsare available as reactive groups or adsorptive groups for the ligand tobind to. These particles are the affinity particles described in claim2, i.e. the particles that are organic particles directly having ontheir surface the phosphorylcholine groups represented by formula (1)and reactive groups or adsorptive groups to which the ligand can bond.When the ligand is bonded to these reactive or adsorptive groups towhich the ligand can bind, the affinity particles described in claim 3,i.e. the particles directly having the phosphorylcholine grouprepresented by formula (1) and the ligand on their surface, areobtained.

The product form of the affinity particles described in claim 2 is suchthat the user can bond any ligand to them depending on the substance tobe captured (target substance). The product form of the affinityparticles described in claim 3 is such that the ligand is alreadybonded. The affinity particles described in claim 1 are affinityparticles having at least the phosphorylcholine group of formula (1) onthe particle surface and their product form is such that the user canbind any ligand to them depending on the substance to be captured(target substance), regardless of the presence or absence of the ligandor reactive group or adsorptive group that can bind to the ligand.Affinity particles of any form are included as long as thephosphorylcholine group of formula (1) is present on the particlesurface; for example, the forms described in claim 2 and claim 3 areincluded as well.

In the aforementioned reaction, leaving some carboxyl groups as reactivegroups or adsorptive groups to which the ligand can bind can be madepossible, for example, by adjusting the reaction quantity of the silanecoupling agent having a carboxylic acid to which the phosphorylcholinegroup is introduced.

It is also possible to react this carboxyl group with a compound havingany functional group and use this functional group as the reactive groupor adsorptive group to which the ligand can bind.

“A Method of Binding the Ligand to the Particles having Carboxyl Groups”

When the ligand is a protein, organic particles having carboxyl groupson the surface are soaked in a solution of N-hydroxysuccinimide and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to esterify the particlesurface. Amino groups in the protein are reacted with these to bind theprotein. It is also possible to react this hydroxyl group with acompound having any functional group and use this functional group asthe reactive group or adsorptive group to which the ligand can bind.

“A Method of Affinity Separation of a Target Substance”

Using the affinity particles of the present invention obtained asdescribed above, the affinity separation of a target substance of thepresent invention is carried out.

The method of the present invention is a groundbreaking separationmethod for a target substance in that high precision separation can beeasily done by using organic particles.

The method of the present invention contains the following 3 processes.The first process is omitted for the affinity particles to which theligand is already bonded (claim 2) since this process has already beendone for such particles.

1. The first process in which any ligand is chemically bonded toaffinity particles that are characterized by having phosphorylcholinegroups represented by the following formula (1) covalently bonded ontothe surface of organic particles or affinity particles that arecharacterized by having phosphorylcholine groups represented by thefollowing formula (1) covalently bonded onto the surface of organicparticles and also by having reactive groups or adsorptive groups, thatare capable of bonding with ligands having specific affinity with acertain target substance, covalently bonded or adsorbed onto the surfaceof organic particles.

For example, 1 ml of a PBS solution of any ligand and affinity particlesthat are organic particles having the phosphorylcholine grouprepresented by formula (1) covalently bonded onto their surface andreactive groups or adsorptive groups to which the ligand can bindcovalently bonded or adsorbed on their surface are put into a 2 ml eppentube, followed by gentle shaking at 4° C. for 30 minutes. This iscentrifuged for 30 minutes at 15,000 rpm and the supernatant isdiscarded. The sample is cleaned by adding 1 ml of a PBS solution to it,gently shaking it, centrifuging it for 30 minutes at 15,000 rpm, anddiscarding the supernatant. This cleaning operation is repeated 3 times.

2. The second process in which the affinity particles prepared in thefirst process are dispersed in a liquid sample containing the targetsubstance that is selectively captured by any ligand.

For example, the affinity particles prepared in the first process aredispersed in a liquid sample containing the target substance that isselectively captured by any ligand, followed by gentle shaking for 30minutes at 4° C. This is centrifuged for 30 minutes at 15,000 rpm andthe supernatant is discarded. The sample is cleaned by adding 1 ml of aPBS solution to it, gently shaking it, centrifuging it for 30 minutes at15,000 rpm, and discarding the supernatant. This cleaning operation isrepeated 3 times.

3. The third process in which the captured target substance is recoveredfrom the separated affinity particles.

For example, for the purpose of recovering the captured target substancefrom the affinity particles, 1 ml of an elution buffer is added,followed by gentle shaking for 30 minutes at 4° C. to elute the targetsubstance from the particles, and the supernatant is recovered. 1 ml ofa PBS solution is added to it, followed by gentle shaking andcentrifugation for 30 minutes at 15,000 rpm, and the supernatant isrecovered. This operation is repeated twice.

FIG. 1 is a schematic showing the differences between the targetsubstance capture selectivity of the affinity particles of the presentinvention and conventional affinity particles.

EXAMPLES

Next, the present invention is described in detail by referring toExamples. The present invention is not limited to these Examples. Thephosphorylcholine group introduced onto the particle surface can beverified and quantified by means of the following method.

<Quantification Method>

The obtained particles were immersed in perchloric acid and heated up to180° C. to be decomposed. The obtained solution was diluted with water,to which hexaammonium heptamolybdate tetrahydrate and L-ascorbic acidwere added, followed by 5 minutes at 95° C. of color development time;the amount introduced was determined by means of the light absorptionmeasurement at 710 nm. For the calibration curve, a sodium dihydrogenphosphate solution was used.

“Synthesis Example 1”

“An Aldehyde Chemical Compound Containing Phosphorylcholine Groups”

1-α-glycerophosphorylcholine (6.29 g) was dissolved in 210 ml ofdistilled water and cooled in an ice water bath. Sodium periodate (10.23g) was added, followed by five hours of stirring. The reaction fluid wasconcentrated under reduced pressure and dried under reduced pressure;methanol was then used to extract the target substance. The structure ofthe compound is shown in the following chemical formula (6).

A 1H NMR spectrum of the compound of formula (6) is shown in FIG. 2.Since the compound of formula (6) is in equilibrium with formula (10) inwater, the actual spectrum reflects both formula (6) and formula (10).

“Synthesis Example 2”

“A Carboxylic Acid Chemical Compound Containing PhosphorylcholineGroups”

5 g of 1-α-glycerophosphorylcholine was dissolved in water (70ml)/acetonitrile (30 ml).

As the temperature was lowered with ice, 17 g of sodium periodate and 80mg of ruthenium trichloride were added, followed by overnight stirring.After filtering the precipitate, concentration under reduced pressureand methanol extraction were carried out to obtain the targetcarboxymethyl phosphorylcholine represented by chemical formula (7).

A 1H NMR spectrum of the compound of formula (7) is shown in FIG. 3.

“Reference Example 1”

“Styrene-Glycidyl Methacrylate Particles”

3.6 g glycidyl methacrylate, 2.4 g styrene, and 0.08 g divinylbenzenewere added to 220 ml of purified water that had been thoroughlydeaerated by means of nitrogen substitution. 0.12 g of polymerizationinitiator V-60 was added, followed by stirring at 70° C. for 1 hour. Anadditional 0.6 g of glycidyl methacrylate was added, followed byovernight stirring at 70° C. After cooling the temperature down to roomtemperature and purification by means of centrifugation (15,000 rpm×30minutes, 3 times), the target particles were obtained.

“Styrene-Glycidyl Methacrylate Particles with Amino Groups Introduced”

1 g of styrene-glycidyl methacrylate particles were dispersed in 80 mlof purified water, to which 20 ml of 25% ammonia aqueous solution wasadded, followed by overnight heated stirring at 70° C. The mixture wascooled down to room temperature and purified by means of centrifugation(17,000 rpm×60 minutes, 3 times).

“Phosphorylcholine-Modified Particles (PC Particles (A))”

0.5 g of styrene-glycidyl methacrylate particles with amino groupsintroduced onto them were dispersed in 10 ml of methanol, to which 0.5of the aldehyde derivative of Synthetic example 1 was added, followed byovernight stirring. 140 mg of sodium cyanoborate was added to themixture in an ice bath, followed by stirring for 6 hours andpurification by means of centrifugation (17,000 rpm×60 minutes, 3 times)to obtain the PC particles (A).

“Agglutination by using Salt”

FIG. 4 and FIG. 5 show the particle size distribution of the PCparticles and styrene-glycidyl methacrylate particles prepared inReference example 1 in water and also in a saline solution (0.1 Maqueous solution).

FIG. 4 shows that styrene-glycidyl methacrylate particles generally usedfor the latex agglutination method using affinity particles have asignificantly different particle size distribution in NaCl solutioncompared with that in water, which indicates that agglutination istaking place. On the other hand, FIG. 5 shows that the particle sizechange of the PC particles (A) in a saline solution is smaller comparedwith that in FIG. 4, indicating that agglutination due to salt occursless. This indicates that the PC particles (A), due to modification withthe phosphorylcholine of formula (1), are less influenced by interferingsubstances such as salt, which leads to a higher measurement accuracy.

“Evaluation of Suppression of Non-Specific Adsorption of Proteins”

25 mg each of the styrene-glycidyl methacrylate particles and PCparticles (A) prepared in Reference example 1 were sampled, to each ofwhich 1 ml of distilled water was added, followed by 1 minute of anultrasonic treatment. After removing distilled water by means ofcentrifugation, 1 mL of albumin (100 μg/mL) or lysozyme (100 μg/mL) wasadded, followed by a 1 hour reaction at room temperature; aftercentrifugation (5,000 g), the supernatant was quantified with the MicroBCA method. The results are shown in FIG. 6. The PC particles (A), whichhad been treated with phosphorylcholine groups, showed significantlysuppressed adsorption of both albumin and lysozyme compared with thestyrene-glycidyl methacrylate particles. This indicates that themodification with the phosphorylcholine of formula (1) significantlyreduces protein adsorption. Not only agglutination between particles butalso the non-specific adsorption of various proteins onto the particlessignificantly contribute to a reduction in the measurement accuracy;therefore the affinity particles of the present invention have asuperior accuracy in selectively capturing only the target protein byusing the ligand.

“Reference Example 2”

“2-aminoethyl methacrylate-N-isopropyl acrylamide-MethyleneBisacrylamide Particles”

1.35 g of N-isopropyl acrylamide and 58 mg of methylene bisacrylamidewere added to 200 ml of purified water thoroughly deaerated by means ofnitrogen substitution. 7 mg of polymerization initiator V-50 was added,followed by stirring at 70° C. for 30 minutes. 100 mg of 2-aminoethylmethacrylate was added to the mixture, followed by additional stirringat 70° C. for 4 hours; after the temperature was cooled down to roomtemperature, it was dialyzed in water and lyophilized to obtain thetarget particles.

“Phosphorylcholine-Modified Particles (PC Particles (B))”

0.1 g of the 2-aminoethyl methacrylate-N-isopropyl acrylamide-methylenebisacrylamide particles were dispersed in 20 ml of methanol, to which 25mg of the aldehyde derivative of Synthetic example 1 was added, followedby overnight stirring. 6 mg of sodium cyanoborate was added in an icewater bath, followed by 6 hours of stirring and purification by means ofdialysis in water to obtain PC particles (B).

“Phosphorylcholine-Modified Particles (PC Particles (C))”

0.1 g of the obtained 2-aminoethyl methacrylate-N-isopropylacrylamide-methylene bisacrylamide particles were dispersed indimethylsulfoxide (8 ml)-water (2 ml) and 1 ml of water in which 25 mgof the carboxyl derivative of Synthesis example 2, 20 mg ofN-hydroxysuccinimide and 23 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved was added, followed byovernight stirring. The mixture was purified by means of dialysis inwater to obtain PC particles (C).

“Evaluation of Suppression of Non-Specific Adsorption of Proteins”

25 mg each of the styrene-glycidyl methacrylate particles prepared inReference example 1 and PC particles (B) and (C) prepared in Referenceexample 2 were sampled, to each of which 1 ml of distilled water wasadded, followed by 1 minute of an ultrasonic treatment. After removingdistilled water by means of centrifugation, 1 mL of albumin (100 μg/mL)or lysozyme (100 μg/mL) was added, followed by a 1 hour reaction at roomtemperature; after centrifugation (5,000 g), the supernatant wasquantified with the Micro BCA method. The results are shown in FIG. 7.The PC particles (B) and (C), which had been treated withphosphorylcholine groups, showed significantly suppressed adsorption ofboth albumin and lysozyme compared with the styrene-glycidylmethacrylate particles. This indicates that the modification with thephosphorylcholine of formula (1) significantly reduces proteinadsorption. Not only agglutination between particles but also thenon-specific adsorption of various proteins onto the particlessignificantly contribute to a reduction in the measurement accuracy;therefore the affinity particles of the present invention have asuperior accuracy in selectively capturing only the target protein byusing the ligand.

“Reference Example 3”

“Phosphorylcholine-Modified Particles (PC Particles (D))”

100 mg of agarose beads (crosslinking ratio 6%) were dispersed in 10 mlof N,N′-dimethylformamide, to which a solution prepared by dissolvingand reacting 50 mg of the carboxyl derivative of Synthetic example 2 and25 mg of thionyl chloride in 1 ml anhydrous N,N′-dimethylformamide wasadded, followed by 3 hours of stirring at room temperature. Purificationby means of successive centrifugation processes first usingN,N′-dimethylformamide and then using acetonitrile was done to obtain PCparticles (D).

“Evaluation of Suppression of Non-Specific Adsorption of Proteins”

25 mg each of the styrene-glycidyl methacrylate particles prepared inReference example 1 and PC particles (D) prepared in Reference example 3were sampled, to each of which 1 ml of distilled water was added,followed by 1 minute of an ultrasonic treatment. After removingdistilled water by means of centrifugation, 1 mL of albumin (100 μg/mL)or lysozyme (100 μg/mL) was added, followed by a 1 hour reaction at roomtemperature; after centrifugation (5,000 g), the supernatant wasquantified with the Micro BCA method. The results are shown in FIG. 8.The PC particles (D), which had been treated with phosphorylcholinegroups, showed significantly suppressed adsorption of both albumin andlysozyme compared with the agarose particles. This indicates that themodification with the phosphorylcholine of formula (1) significantlyreduces protein adsorption. Not only agglutination between particles butalso the non-specific adsorption of various proteins onto the particlessignificantly contribute to a reduction in the measurement accuracy;therefore the affinity particles of the present invention have asuperior accuracy in selectively capturing only the target protein byusing the ligand.

“Example 1”

“Affinity Particles”

Next, the affinity separation method shown in claim 6 is described. 10mg of the aldehyde derivative of Synthetic example 1 was added to 0.1 gof the 2-aminoethyl methacrylate-N-isopropyl acrylamide-methylenebisacrylamide particles obtained in Reference example 2, followed byovernight stirring, after which 3 mg of sodium cyanoborate was added tothe mixture in an ice water bath, followed by 6 hours of stirring anddialysis in water to obtain the affinity particles. 1 mL of aglutaraldehyde solution (8%) and 10 mg of sodium cyanotrihydroborate,for stabilizing Schiff base, were added to these affinity particles andthe reaction was carried out for 5 hours at room temperature, followedby 5 times of a centrifugation/purification (5,000 g) operation usingPBS for cleaning The affinity particles of claim 2 that haveglutaraldehyde as reactive groups or adsorptive groups to which theligand can bind were thus obtained. 1 mL of bovine albumin (1 mg/mL) orhuman hemoglobin (1 mg/mL) and 10 mg of sodium trihydroborate were addedand the reaction was carried out for 1 day at room temperature, followedby 4 times of a centrifugation/purification (5,000 g) with PBS. Thisbovine albumin or human hemoglobin is the ligand. After this is theaffinity separation method shown in claim 7. 1 mL of ethanolaminehydrochloride (0.5 M, pH 7.1) and 10 mg of sodium trihydroborate wereadded and the reaction was carried out for 1 hour at room temperature todeactivate the remaining glutaraldehyde, followed by 4 times of acentrifugation/purification (5,000 g) with PBS to obtain the affinityparticles of claim 3. 1 mL of HRP-conjugated anti-bovine albuminantibody (10 μg/mL) or HRP-conjugated human hemoglobin antibody (10μg/mL) were added and the reaction was carried out for 1 hour at roomtemperature, followed by 5 times of a centrifugation/purification (5,000g) with PBS. An additional 1 mL of PBS was added, followed by stirring;10 μl each was transferred onto a 96-hole well plate and a colordevelopment test was conducted using substrate TMBZ; the measurement wasdone at 450 nm. The results are shown in FIG. 9. The target antibody wascaptured in a highly selective manner for either ligand used.

Comparative Example 1

FIG. 10 shows the result of the same operation as Example except for thefact that 0.1 g of the 2-aminoethyl methacrylate-N-isopropylacrylamide-methylene bisacrylamide particles obtained in Referenceexample 2 were not modified with phosphorylcholine. The selectivity wasshown to be lower compared with Example 1 for either ligand.

INDUSTRIAL APPLICABILITY

The affinity particles of the present invention capture only the targetsubstance that is desired to be separated and therefore they exhibitvery high selectivity. They also exhibit superior dispersion propertiesand make separation from liquid samples very easy. Also, lessagglutination caused by salts means easy and highly accurate separationof the target substance. Also, they are useful as reagents for theimmunoprecipitation method and the latex agglutination method inbio-industries where a highly accurate separation and detection of thetarget substance is required since they are immune to the influence ofsalts and capable of highly sensitive detection.

1. Affinity particles comprising phosphorylcholine groups represented bythe following formula (1), covalently bonded onto the surface of organicparticles.


2. Affinity particles comprising phosphorylcholine groups represented bythe following formula (1), covalently bonded onto the surface of organicparticles, and also by having reactive groups or adsorptive groups,which are capable of bonding with ligands having specific affinity witha certain target substance, covalently bonded or adsorbed onto thesurface of organic particles.


3. Affinity particles comprising phosphorylcholine groups represented bythe following formula (1), covalently bonded onto the surface of organicparticles, and also by having ligands having specific affinity with acertain target substance covalently bonded or adsorbed onto the surfaceof organic particles.


4. The affinity particles of claim 1, organic particles are eithersynthetic particles whose polymers contain one, two, or more types ofmonomer units chosen from a group consisting of styrene, glycidylmethacrylate, (meth)acrylic acid, N-alkylacrylamide, and alkyl(meth)acrylate, or polysaccharides composed of agarose or sepharosehaving an average particle size of 20 nm to 500 μm.
 5. The affinityparticles of claim 2, wherein said ligands are one, two, or more typesof ligands chosen from a group consisting of various antibodies,antigens, enzymes, substrates, receptors, peptides, DNA, RNA, aptamers,protein A, protein G, avidin, biotin, chelating compounds, and variousmetal ions.
 6. A method of affinity separation of a target substance byusing organic particles comprising: (1) a first process wherebyarbitrary ligands are bonded to the affinity particles of claim 1, (2) asecond process whereby the affinity particles prepared in the firstprocess are dispersed in a liquid sample containing a target substanceselectively captured by the arbitrary ligands, and (3) a third processwhereby the target substance captured is recovered from the affinityparticles.
 7. A method of affinity separation of a target substance byusing organic particles comprising: (1) a first process whereby theaffinity particles of claim 3 are dispersed in a liquid samplecontaining a target substance selectively captured by the arbitraryligands, and (2) a second process whereby the target substance capturedis recovered from the affinity particles.
 8. The affinity particles ofclaim 2, wherein said organic particles are either synthetic particleswhose polymers contain one, two, or more types of monomer units chosenfrom a group consisting of styrene, glycidyl methacrylate, (meth)acrylicacid, N-alkylacrylamide, and alkyl (meth)acrylate, or polysaccharidescomposed of agarose or sepharose having an average particle size of 20nm to 500 μm.
 9. The affinity particles of claim 3, wherein said organicparticles are either synthetic particles whose polymers contain one,two, or more types of monomer units chosen from a group consisting ofstyrene, glycidyl methacry late, (meth)acrylic acid, N-alkylacrylamide,and alkyl (meth)acrylate, or polysaccharides composed of agarose orsepharose having an average particle size of 20 nm to 500 μm.
 10. Theaffinity particles of claim 3, wherein said ligands are one, two, ormore types of ligands chosen from a group consisting of variousantibodies, antigens, enzymes, substrates, receptors, peptides, DNA,RNA, aptamers, protein A, protein G, avidin, biotin, chelatingcompounds, and various metal ions.
 11. The affinity particles of claim4, wherein said ligands are one, two, or more types of ligands chosenfrom a group consisting of various antibodies, antigens, enzymes,substrates, receptors, peptides, DNA, RNA, aptamers, protein A, proteinG, avidin, biotin, chelating compounds, and various metal ions.
 12. Amethod of affinity separation of a target substance by using organicparticles comprising: (1) a first process whereby arbitrary ligands arebonded to the affinity particles of claim 2, (2) a second processwhereby the affinity particles prepared in the first process aredispersed in a liquid sample containing a target substance selectivelycaptured by the arbitrary ligands, and (3) a third process whereby thetarget substance captured is recovered from the affinity particles.